The present invention is in the field of animal health, and is directed to vaccine compositions and diagnostics for disease. More particularly, the present invention relates to polynucleotide molecules that can be used as vaccine components against feline immunodeficiency virus.
Feline immunodeficiency virus (FIV) infection in cats results in a disease syndrome similar to that caused in humans by human immunodeficiency virus-1 (HIV-1) infection. After infection of cats by FIV, disease progression begins with a transient acute phase illness (8 to 10 weeks), followed by a prolonged asymptomatic phase varying from weeks to years, and a terminal symptomatic phase (Ishida and Tomoda, 1990, Jpn. J. Vet Sci. 52:645-648; English et al., 1994, J. Infect. Dis. 170: 543-552). Similar to HIV-1 disease progression (Graziosi et al., 1993, Proc. Natl. Acad. Sci. 90:6405-6409; Baumberger et al., 1993, AIDS 7:S59-S64; Wei et al., 1995, Nature 373:117-122), FIV RNA load in plasma has been demonstrated to correlate with disease stage, and can predict disease progression in accelerated FIV infection (Diehl et al., 1995, J. Virol. 69:2328-2332; Diehl et al., 1996, J. Virol. 70:2503-2507).
Based on the genetic diversity of the ENV protein of FIV, especially the V3 region, five FIV subtypes have been proposed: subtypes A and B, mainly in North America, Europe and Japan; subtype C in British Columbia and Taiwan; subtype D in Japan; and subtype E in Argentina (Sodora et al., 1994, J. Virol. 68:2230-2238; Kakinuma et al., 1995, J. Virol. 69:3639-3646; and Pecoraro et al., 1996, J. Gen. Virol. 77:2031-2035).
Similar to other lentiviruses, such as HIV-1, the FIV genome contains three large open reading frames, i.e., GAG (group antigens), ENV (envelope), and POL (polymerase), and three small open reading frames encoding regulatory (i.e., non-structural) proteins, i.e., Rev (regulator of expression of virion protein), Vif (virion infectivity factor) and ORF2 (open reading frame 2). The provirus contains two long terminal repeats (LTR), one at each end of the genome (Talbott et al., 1989, Proc. Natl. Acad. Sci. USA 86:5743-5747; Olmsted et al., 1989, Proc. Natl. Acad. Sci. USA 86:8088-8092). GAG is a precursor polyprotein that is processed into three mature virion structural proteins, i.e., the matrix (MA), capsid (CA) and nucleocapsid (NC) proteins. ENV is a precursor protein that is processed into two envelope structural proteins, i.e., the surface (SU) and transmembrane (TM) proteins. POL encodes four enzymatic (i.e., non-structural) proteins, i.e., protease (PR), reverse transcriptase (RT), deoxyuridine triphosphatase (DU) and integrase (IN).
The mechanism by which protective immunity against FIV infection can be achieved remains poorly understood. It has been reported by some groups that virus neutralizing (VN) antibodies appear to play a major role in the observed protection (Yamamoto et al., 1991, AIDS Res. Hum. Retrovir. 7:911-922; Hosie et al., 1995, J. Virol. 69:1253-1255). Consistent with those observations was the finding that cats who passively received antibodies from vaccinated or infected cats were protected from homologous challenge (Hohdatsu et al., 1993, J. Virol. 67:2344-2348; Pu et al., 1995, AIDS 9:235-242).
By contrast, convincing data also indicates that the levels of antibodies, or even VN antibodies, do not correlate with protection. It has been reported that cats were protected against homologous challenge in the absence of detectable VN antibodies (Verschoor et al., 1995, Vet. Immunol. Immunopathol. 46:139-149; Matteucci et al., 1996, J. Virol. 70:617-622). In addition, other vaccinated cats failed to be protected in the presence of significant VN antibodies (Huisman et al., 1998, Vaccine 16:181-187; Flynn et al., 1997 J. Virol. 71:7586-7592; Tijhaar et al., 1997, Vaccine 15:587-596; Osterhaus et al., 1996, AIDS Res. Hum. Retrovir. 12:437-441; Verschoor et al., 1996, Vaccine 14:285-289; Rigby et al., 1996, Vaccine 14:1095-1102; Lutz et al., 1995, Vet. Immunol. Immunopathol. 46:103-113; Flynn et al., 1995, Immunol. 85:171-175; Gonin et al., 1995, Vet. Microbiol. 45:393-401). This discrepancy appears to result, at least partially, from the different cell systems and virus isolates used in the VN assays. It has recently become evident that fresh isolates of FIV obtained from naturally infected cats are much less sensitive to VN antibodies than laboratory viruses adapted to growth in tissue culture (Baldinotti et al., 1994, J. Virol. 68: 4572-4579). It has also been found that the same antibodies which neutralized FIV infection in Crandell Feline Kidney (CRFK) cells failed to neutralize FIV infection in primary feline thymocytes (Huisman et al., 1998, above). These data indicate that the VN antibodies detected in vitro may not play any role in protective immunity in vivo.
In a few limited reports, cell-mediated immunity was investigated following vaccination. In one report, it was found that cellular immunity, especially ENV-specific CTL responses, played a major role in protecting cats vaccinated with whole inactivated virus (Flynn et al., 1996, J. Immunol. 157:3658-3665; Flynn et al., 1995, AIDS Res. Hum. Retrovir. 11:1107-1113). It was also reported that long-term protection was more closely correlated with the induction of ENV-specific cytotoxic T-cell activity (Hosie and Flynn, 1996, J. Virol. 70:7561-7568).
It appears that both humoral and cellular immunity are involved in achieving protective immunity in the acute phase after challenge, but for long-term protection, cell-mediated immunity appears to be more important. However, the question still remains which viral protein(s) or subunit(s) or combinations are capable of inducing protective immune responses. In one report, although both cell-mediated and humoral immune responses were induced in cats vaccinated with a multi-epitopic peptide within the ENV protein, vaccination did not confer protection against homologous challenge (Flynn et al., 1997, above).
As in HIV-1, an observation that complicates the development of an effective FIV vaccine is the enhancement of infection that has been observed in cats immunized with certain vaccines. Such enhancement of infection has been observed in a number of FIV vaccine trials in which either recombinant subunit vaccines, synthetic vaccines, whole inactivated virus vaccines or fixed, infected cell vaccines were used to vaccinate cats (Osterhaus et al., 1996, above; Siebelink et al., 1995, J. Virol. 69:3704-3711; Lombardi et al., 1994, J. Virol. 68:8374-8379; Hosie et al., 1992, Vet. Immunol. Immunopathol. 35:191-197; Huisman et al., 1998, above). For example, in an ENV subunit vaccine trial, enhancement of infection occurred despite anti-ENV and VN antibody production, and this enhancement could be transferred to naive cats via plasma pools from the vaccinated animals, indicating that the enhancement was probably mediated by specific antibodies (Siebelink et al., 1995, above).
It appears that antibodies against ENV tend to enhance infection more readily than antibodies against GAG protein. However, the mechanism by which antibodies enhance FIV infection remains poorly understood. In HIV-1, antibody-dependent enhancement requires that the target cells express either the immunoglobulin Fc receptor (FcR), or complement receptors (CRs). The enhancement is a biphasic response based on serum dilution; that is, at higher antibody concentrations, viral neutralization is observed, whereas enhancement is seen at lower antibody concentrations (Mascola et al., 1993, AIDS Res. Hum. Retrovir. 9:1175-1184). The enhanced infectivity may interfere with the induction of protective immunity in FIV , which may partially explain the reason why a large number of FIV vaccination experiments in which ENV protein or its subunits were used as vaccines were unsuccessful. Therefore, the rational development of vaccines against lentiviruses, including FIV and HIV-1, requires the careful assessment and selection of vaccine immunogens.
Since the discovery of FIV, many attempts have been made to develop a safe and effective FIV vaccine. Three different groups have attempted to vaccinate cats with fixed virus-infected cells; however, conflicting results were obtained from these vaccination trials. The first group found that all the cats vaccinated with fixed FIV-infected cells were protected from challenge with plasma obtained from cats infected with the homologous virus, despite the fact that no VN antibodies were detected after vaccination (Matteucci et al., 1996, above). The protection conferred by this vaccine, however, was relatively short-lived and difficult to boost (Matteucci et al., 1997, J. Virol. 71:8368-8376). Similar results were reported by the second group describing protection against homologous, but not heterologous, FIV challenge up to 12 weeks post-challenge (Bishop et al., 1996, Vaccine 14:1243-1250). However, when cats were monitored up to week 50 post-challenge, a loss of protection against the homologous virus was observed. Also, protection could not be correlated with the levels of antibody to p24 capsid protein or VN titers. In contrast to these findings, the third group reported no protection when ten cats were vaccinated with a fixed FIV-infected cell vaccine. Eight of the cats became viraemic 5 weeks post-challenge, although significant VN antibodies were detected at the time of challenge (Verschoor et al., 1995, above).
Another type of conventional FIV vaccine that has been tested is whole, inactivated virus. The first successful whole-inactivated FIV vaccine was reported by Yamamoto""s group, which observed greater than 90% protection against homologous challenge (Yamamoto et al., 1991, AIDS Res. Hum. Retrovir. 7:911-922), and slight protection against heterologous challenge (Yamamoto et al., 1993, J. Virol. 67:601-605). Both humoral and cellular immunity against FIV were induced and high levels of anti-ENV, anti-core, and VN antibodies were observed in the vaccinated cats. Recent studies have indicated that both virus-specific humoral immunity, especially VN antibodies, and cellular immunity, especially the ENV-specific CTL responses, play a role in the protection induced in cats vaccinated with whole, inactivated virus (Hosie and Flynn, 1996, above; Flynn et al., 1996, above; Hosie et al., 1995, above; Elyar et al., 1997, Vaccine 15:1437-1444). However, in contrast to the studies described above, vaccination of cats with whole, inactivated FIV incorporated into immune stimulating complexes (ISCOMS) failed to protect against homologous challenge (Hosie et al., 1992, above).
Another approach for FIV vaccine development that has been extensively investigated recently is recombinant vaccines. A number of FIV subunit vaccines have been tested, including those containing recombinant core protein, synthetic V3, or multi-epitopic peptides, glycosylated or unglycosylated recombinant ENV protein, and various vector-based systems (Elyar et al., 1997, above). Unfortunately, although significant levels of antibodies were generally induced by such vaccinations, all attempts failed to protect vaccinated cats against homologous challenge (Huisman et al., 1998, above; Flynn et al., 1997, above; Tijhaar et al., 1997, above; Osterhaus et al., 1996, above; Verschoor et al., 1996, above; Rigby et al., 1996, above; Lutz et al., 1995, above; Flynn et al., 1995, Immunol. 85:171-175; Gonin et al., 1995, above).
Recently, a DNA vaccine was tested for FIV. Cats vaccinated with plasmid DNA carrying FIV structural genes, including ENV and p10 gene (i.e., the NC protein of FIV), exhibited strong humoral immune responses. However, none of the vaccinated cats were protected from homologous challenge (Cuisinier et al., 1997, Vaccine 15: 1085-1094).
In addition, WO 98/03660 describes various formulae for feline polynucleotide vaccines including against FIV, but only mentions the use of ENV polyprotein and GAG/PRO polyprotein genes, and does not describe the use of other FIV genes, or substituent genes from the particular polyprotein genes, nor does it provide any data showing efficacy of any particular FIV vaccine.
The present invention provides a vaccine composition against feline immunodeficiency virus (FIV), comprising an immunologically effective amount of a polynucleotide molecule comprising a nucleotide sequence selected from a portion of the genome of an FIV strain, or a nucleotide sequence which is a degenerate variant thereof; and a veterinarily acceptable carrier. The FIV strain can be any strain of FIV, but is preferably strain FIV-141 having a genomic RNA sequence corresponding to the DNA sequence shown in SEQ ID NO:1 from nt 1 to nt 9464.
In a preferred embodiment, the polynucleotide molecule of the vaccine composition comprises a nucleotide sequence encoding one or more of a structural or non-structural protein from an FIV strain, or a combination thereof. The structural protein is selected from the group consisting of a GAG protein and an ENV protein. The non-structural protein is selected from the group consisting of a POL protein and a regulatory protein. The GAG protein is selected from the group consisting of the GAG polyprotein and its substituent proteins, i.e., MA, CA and NC. The ENV protein is selected from the group consisting of the ENV polyprotein and its substituent proteins, i.e., SU and TM. The POL protein is selected from the group consisting of the POL polyprotein and its substituent proteins, i.e., PR, RT, DU and IN. The regulatory protein is selected from the group consisting of Rev, Vif and ORF2.
The polynucleotide molecule of the vaccine composition may alternatively or additionally comprise a nucleotide sequence consisting of a substantial portion of any of the aforementioned nucleotide sequences. In a preferred embodiment, the substantial portion of the nucleotide sequence encodes an epitope of an FIV protein.
In a preferred embodiment, the vaccine composition of the present invention comprises a polynucleotide molecule comprising a nucleotide sequence encoding an FIV protein selected from the group consisting of GAG, MA, CA, NC, ENV, SU, TM, DU and PR.
In a more preferred embodiment, the vaccine composition of the present invention is a combination vaccine, which comprises one or more polynucleotide molecules having nucleotide sequences encoding a combination of FIV proteins. In a preferred embodiment, the one or more polynucleotide molecules of the vaccine composition comprise nucleotide sequences encoding at least two different FIV proteins selected from FIV structural and FIV non-structural proteins, provided that when the one or more polynucleotide molecules encode the ENV and NC proteins from FIV, they also encode at least one, preferably at least two, and most preferably at least three other FIV structural or non-structural proteins.
In a further preferred embodiment, the one or more polynucleotide molecules of the vaccine composition comprise nucleotide sequences encoding at least two different GAG proteins from FIV.
In a further preferred embodiment, the one or more polynucleotide molecules of the vaccine composition comprise nucleotide sequences encoding at least one FIV structural protein and at least one FIV non-structural protein.
In a further preferred embodiment, the one or more polynucleotide molecules of the vaccine composition comprise nucleotide sequences encoding at least three different FIV proteins selected from among the FIV structural and FIV non-structural proteins, i.e., the proteins can be either all structural proteins or all non-structural proteins, or a combination of structural and non-structural proteins. In a further preferred embodiment, the one or more polynucleotide molecules of the vaccine composition comprise nucleotide sequences encoding at least four different FIV proteins selected from among the FIV structural and FIV non-structural proteins. In a further preferred embodiment, the one or more polynucleotide molecules of the vaccine composition comprise nucleotide sequences encoding at least five different FIV proteins selected from among the FIV structural and FIV non-structural proteins. In a further preferred embodiment, the one or more polynucleotide molecules of the vaccine composition comprise nucleotide sequences encoding at least six different FIV proteins selected from among the FIV structural and FIV non-structural proteins. In a further preferred embodiment, the one or more polynucleotide molecules of the vaccine composition comprise nucleotide sequences encoding at least seven different FIV proteins selected from among the FIV structural and FIV non-structural proteins.
In a further preferred embodiment, the one or more polynucleotide molecules of the vaccine composition comprise a nucleotide sequence encoding at least one FIV structural protein and a nucleotide sequence encoding at least one FIV regulatory protein. In a further preferred embodiment, the one or more polynucleotide molecules of the vaccine composition comprise a nucleotide sequence encoding at least one FIV POL protein and a nucleotide sequence encoding at least one FIV regulatory gene. In a further preferred embodiment, the one or more polynucleotide molecules of the vaccine composition comprise a nucleotide sequence encoding at least one FIV structural protein, a nucleotide sequence encoding at least one FIV POL protein, and a nucleotide sequence encoding at least one FIV regulatory protein.
In a further preferred embodiment, when the one or more polynucleotide molecules of the vaccine composition comprise nucleotide sequences encoding a GAG protein, PR protein or ENV protein from FIV, or a combination thereof, one or more nucleotide sequences encoding at least one, more preferably at least two, and most preferably at least three other FIV proteins are present.
In a particularly preferred embodiment, the vaccine composition of the present invention comprises one or more polynucleotide molecules comprising nucleotide sequences encoding a combination of FIV proteins, which combination is selected from the group consisting of GAG/MA/CA/NC; GAG/ENV; GAG/MA/CA/NC/ENV/SU/TM; MA/CA/NC; GAG/MA/NC/DU/PR; and MA/CA/NC/SU/TM. When the vaccine composition of the present invention is a combination vaccine, the nucleotide sequences encoding the various FIV proteins or polypeptides can be on the same polynucleotide molecule, on different polynucleotide molecules, or a combination thereof.
The polynucleotide molecule of the vaccine composition can either be a DNA or RNA molecule, although DNA is preferred. The polynucleotide molecule of the vaccine composition is preferably administered as part of an expression vector construct, such as a plasmid or a viral vector.
The present invention further provides a method of preparing a vaccine composition against FIV, comprising combining an immunologically effective amount of any one or more of the aforementioned polynucleotide molecules, or any one or more expression vectors comprising such polynucleotide molecules, with a veterinarily acceptable carrier in a form suitable for administration to cats. In a non-limiting embodiment, a veterinarily acceptable carrier is selected from standard aqueous or partially aqueous solutions, such as sterile saline or PBS, or cationic lipid preparations, or gold microparticles onto which the one or more polynucleotide molecules or expression vectors of the vaccine composition can be coated and administered to an animal for vaccine delivery. The vaccine composition can further comprise a supplemental component such as, e.g., an immunomodulatory agent, which can be an adjuvant, or a cytokine, or a polynucleotide molecule having a nucleotide sequence encoding a cytokine; or an agent which facilitates cellular uptake by the vaccinated animal of the polynucleotide molecule or expression vector; or a combination thereof.
The present invention further provides a method of vaccinating a cat against FIV, comprising administering to the cat a vaccine composition of the present invention. In a preferred though non-limiting embodiment, the vaccine composition of the present invention is administered to a cat either by intramuscular or intradermal injection, or orally, intranasally, or by use of a gene gun.
The present invention further provides a kit for vaccinating a cat against FIV, comprising a first container comprising an immunologically effective amount of any one or more of the aforementioned polynucleotide molecules or expression vectors of the present invention. In a non-limiting embodiment, the one or more polynucleotide molecule or expression vectors are stored in the first container in lyophilized form. The kit may optionally further comprise a second container comprising a sterile diluent useful to dilute or rehydrate the polynucleotide molecules or expression vectors in the first container for administration to a cat.
The present invention further provides an isolated antibody that binds specifically to an FIV protein, which antibody is produced in a mammal in response to administration of a polynucleotide molecule having a nucleotide sequence encoding the FIV protein or an epitope thereof, such as, e.g., a polynucleotide molecule or expression vector as present in the vaccine composition of the present invention.
The present invention further provides a vaccine composition against FIV, comprising an immunologically effective amount of a GAG protein, POL protein, ENV protein, regulatory protein, or a combination thereof, from an FIV strain. The FIV strain can be any strain of FIV, but is preferably strain FIV-141. The GAG protein is preferably selected from the group consisting of the GAG polyprotein and its substituent proteins, i.e., MA, CA and NC. The POL protein is preferably selected from the group consisting of the POL polyprotein and its substituent proteins, i.e., PR, RT, DU and IN. The ENV protein is preferably selected from the group consisting of the ENV polyprotein and its substituent proteins, i.e., SU and TM. The regulatory protein is selected from the group consisting of Rev, Vif and ORF2.
In a more preferred embodiment, the vaccine composition of the present invention comprises a combination of FIV proteins. In a preferred embodiment, the proteins of the vaccine composition comprise at least two different FIV proteins selected from among the FIV structural and FIV non-structural proteins. In a further preferred embodiment, the proteins of the vaccine composition comprise at least two different GAG proteins from FIV. In a further preferred embodiment, the proteins of the vaccine composition comprise at least one FIV structural protein and at least one FIV non-structural protein. In a further preferred embodiment, the proteins of the vaccine composition comprise at least three different FIV proteins selected from among the FIV structural and FIV non-structural proteins. In a further preferred embodiment, the proteins of the vaccine composition comprise at least four different FIV proteins selected from among the FIV structural and FIV non-structural proteins. In a further preferred embodiment, the proteins of the vaccine composition comprise at least five different FIV proteins selected from among the FIV structural and FIV non-structural proteins. In a further preferred embodiment, the proteins of the vaccine composition comprise at least six different FIV proteins selected from among the FIV structural and FIV non-structural proteins. In a further preferred embodiment, the proteins of the vaccine composition comprise at least seven different FIV proteins selected from among the FIV structural and FIV non-structural proteins.
In a particularly preferred embodiment, the combination of FIV proteins is selected from the group consisting of GAG/MA/CA/NC; GAG/ENV; GAG/MA/CA/NC/ENV/SU/TM; MA/CA/NC; GAG/MA/NC/DU/PR; and MA/CA/NC/SU/TM.
Alternatively or additionally, the vaccine composition may comprise one or more polypeptides, one or more of which is a substantial portion of an FIV protein. In a preferred embodiment, the substantial portion of the FIV protein comprises an epitope of an FIV protein.
The vaccine composition of the present invention may alternatively comprise an immunologically effective amount of any one or more of the aforementioned polynucleotide molecules or expression vectors in combination with any one or more of the aforementioned proteins or polypeptides.
The present invention further provides a method of preparing a vaccine composition against FIV, comprising combining an immunologically effective amount of any one or more of the aforementioned proteins or polypeptides with a veterinarily acceptable carrier in a form suitable for administration to cats. The vaccine composition can further comprise a supplemental component such as, e.g., an immunomodulatory agent, which can be an adjuvant, or a cytokine, or a polynucleotide molecule having a nucleotide sequence encoding a cytokine, or a combination thereof.
The present invention further provides a method of vaccinating a cat against FIV, comprising administering to the cat a vaccine composition comprising an immunologically effective amount of any one or more of the aforementioned proteins or polypeptides.
The present invention further comprises oligonucleotide molecules that can be used as primers to specifically amplify particular FIV genes or other FIV-related polynucleotide molecules, and as diagnostic probes to detect the present of an FIV-related polynucleotide molecule in a fluid or tissue sample collected from an animal infected with FIV. In a preferred embodiment, such oligonucleotide molecules comprise nucleotide sequences selected from the group consisting of SEQ ID NOS: 2 to 47, or the complements of said sequences.